Antibodies to matrix metalloproteinase 9

ABSTRACT

The present disclosure provides compositions and methods of use involving binding proteins, e.g., antibodies and antigen-binding fragments thereof, that bind to the matrix metalloproteinase-9 (MMP9) protein (MMP9 is also known as gelatinase-B), wherein the binding proteins comprise an immunoglobulin (Ig) heavy chain (or functional fragment thereof) and an Ig light chain (or functional fragment thereof).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/619,318, filed Sep. 14, 2012, now allowed, which is a divisional ofU.S. patent application Ser. No. 13/219,523, filed Aug. 26, 2011, nowU.S. Pat. No. 8,377,443, which claims priority benefit of U.S.Provisional Patent Application No. 61/377,886, filed Aug. 27, 2010. Thecontents of each of these applications are incorporated herein byreference in their entirety for all purposes.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 246102008001Seqlist.txt,date recorded: Jul. 1, 2013, size: 39,055 bytes).

FIELD

This disclosure is in the field of extracellular enzymes, extracellularmatrix enzymes, proteases and immunology.

INTRODUCTION

Matrix metalloproteinases (MMPs) are a family of extracellular enzymesinvolved in forming and remodeling the extracellular matrix. Theseenzymes contain a conserved catalytic domain in which a zinc atom iscoordinated by three histidine residues. Currently, over 20 members ofthis family are known, organized into a number of groups includingcollagenases, gelatinases, stromelysins, matrilysins, enamelysins andmembrane MMPs.

MMP2 and MMP9 belong to the gelatinase group of matrixmetalloproteinases. Besides containing signal peptide, propeptide,catalytic, zinc-binding and heamopexin-like domains common to most MMPs,the gelatinases also contain a plurality of fibronectin-like domains andan O-glycosylated domain.

Abnormal activity of certain MMPs has been shown to play a role in tumorgrowth, metastasis, inflammation and vascular disease. See, for example,Hu et al. (2007) Nature Reviews Drug Discovery 6:480-498. Because ofthis, it can be desirable to inhibit the activity of one or more MMPs incertain therapeutic settings. However, the activity of certain otherMMPs is often required for normal function. Since most MMP inhibitorsare targeted to the conserved catalytic domain and, as a result, inhibita number of different MMPS, their therapeutic use has caused sideeffects due to the inhibition of essential, non-pathogenically-relatedMMPs.

Despite this problem, it has proven difficult to develop inhibitors thatare specific to a particular MMP, because inhibition of enzymaticactivity generally requires that the inhibitor be targeted to thecatalytic domain. Consequently, most inhibitors of matrixmetalloproteinase enzymatic activity are likely to react with more thanone MMP, due to homologies in their catalytic domains. Thus, thereremains a need for therapeutic reagents that specifically inhibit thecatalytic activity of a single MMP, and that do not react with otherMMPs.

SUMMARY

The present disclosure provides compositions and methods of useinvolving binding proteins, e.g., antibodies and antigen-bindingfragments thereof, that bind to the matrix metalloproteinase-9 (MMP9)protein (MMP9 is also known as gelatinase-B), wherein the bindingproteins comprise an immunoglobulin (Ig) heavy chain (or functionalfragment thereof) and an Ig light chain (or functional fragmentthereof). The disclosure further provides MMP9 binding proteins thatbind specifically to MMP9 and not to other, related matrixmetalloproteinases. Such MMP9 binding proteins find use in applicationsin which it is necessary or desirable to obtain specific modulation(e.g., inhibition) of MMP9, e.g., without directly affecting theactivity of other matrix metalloproteinases. Thus, in certainembodiments of the present disclosure an anti-MMP9 antibody is aspecific inhibitor of the activity of MMP9. In particular, the MMP9binding proteins disclosed herein will be useful for inhibition of MMP9while allowing normal function of other, related matrixmetalloproteinases.

Accordingly, the present disclosure provides, inter alia:

1. A MMP9 binding protein comprising an immunoglobulin heavy chain orfunctional fragment thereof, and an immunoglobulin light chain orfunctional fragment thereof, wherein the protein does not bind to amatrix metalloproteinase other than MMP9.

2. The protein of embodiment 1, wherein the heavy chain comprises acomplementarity-determining region (CDR) selected from one or more ofSEQ ID NOs: 13-15, and the light chain comprises a CDR selected from oneor more of SEQ ID NOs: 16-18.

3. The protein of embodiment 2, wherein the heavy chain comprises avariable region selected from the group consisting of SEQ ID NOs: 3 or5-8, and the light chain comprises a variable region selected from thegroup consisting of SEQ ID NOs: 4 or 9-12.

4. The protein of embodiment 1, wherein the heavy chain is an IgG.

5. The protein of embodiment 1, wherein the light chain is a kappachain.

6. The protein of embodiment 1, wherein the binding of the protein toMMP9 inhibits the enzymatic activity of MMP9.

7. The protein of embodiment 6, wherein the inhibition isnon-competitive.

8. The protein of embodiment 1, wherein the heavy chain is encoded by apolynucleotide having a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 19-22 and the light chain is encoded by apolynucleotide having a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 23-26.

9. A vector comprising one or more polynucleotides having a nucleotidesequence selected from the group consisting of SEQ ID NOs: 19-26.

10. A cell comprising the vector of embodiment 9.

11. A pharmaceutical composition comprising the protein of embodiment 1.

12. A pharmaceutical composition comprising the vector of embodiment 9.

13. A pharmaceutical composition comprising the cell of embodiment 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of the heavy chain variable regionof a mouse monoclonal anti-MMP9 antibody (AB0041), along with the aminoacid sequences of humanized variants of heavy chain (VH1-VH4), alignedto show differences in framework amino acid sequence resulting fromhumanization. CDRs are shown in italics, and amino acids that aredifferent in the humanized variants, compared to the parent mousemonoclonal, are underlined.

FIG. 2 shows the amino acid sequence of the light chain variable regionof a mouse monoclonal anti-MMP9 antibody (AB0041), along with the aminoacid sequences of humanized variants of this light chain (VH1-VH4),aligned to show differences in framework amino acid sequence resultingfrom humanization. CDRs are shown in italics, and amino acids that aredifferent in the humanized variants, compared to the parent mousemonoclonal, are underlined.

FIG. 3 shows a schematic diagram of the MMP9 protein.

DETAILED DESCRIPTION

Practice of the present disclosure employs, unless otherwise indicated,standard methods and conventional techniques in the fields of cellbiology, toxicology, molecular biology, biochemistry, cell culture,immunology, oncology, recombinant DNA and related fields as are withinthe skill of the art. Such techniques are described in the literatureand thereby available to those of skill in the art. See, for example,Alberts, B. et al., “Molecular Biology of the Cell,” 5″ edition, GarlandScience, New York, N.Y., 2008; Voet, D. et al. “Fundamentals ofBiochemistry: Life at the Molecular Level,” 3^(rd) edition, John Wiley &Sons, Hoboken, N.J., 2008; Sambrook, J. et al., “Molecular Cloning: ALaboratory Manual,” 3^(rd) edition, Cold Spring Harbor Laboratory Press,2001; Ausubel, F. et al., “Current Protocols in Molecular Biology,” JohnWiley & Sons, New York, 1987 and periodic updates; Freshney, R. I.,“Culture of Animal Cells: A Manual of Basic Technique,” 4^(th) edition,John Wiley & Sons, Somerset, N.J., 2000; and the series “Methods inEnzymology,” Academic Press, San Diego, Calif.

See also, for example, “Current Protocols in Immunology,” (R. Coico,series editor), Wiley, last updated August 2010.

MMP9 Binding Proteins

The present disclosure provides binding proteins, e.g., antibodies andantigen-binding fragments thereof, that bind to the matrixmetalloproteinase-9 (MMP9) protein (MMP9 is also known as gelatinase-B),The binding proteins of the present disclosure generally comprise animmunoglobulin (Ig) heavy chain (or functional fragment thereof) and anIg light chain (or functional fragment thereof).

The disclosure further provides MMP9 binding proteins that bindspecifically to MMP9 and not to other matrix metalloproteinases such asMMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP12, MMP13. Such specific MMP9binding proteins are thus generally not significantly or detectablycrossreactive with non-MMP9 matrix metalloproteinases. MMP9 bindingproteins that specifically bind MMP9 find use in applications in whichit is necessary or desirable to obtain specific modulation (e.g.,inhibition) of MMP9, e.g., without directly affecting the activity ofother matrix metalloproteinases.

In certain embodiments of the present disclosure an anti-MMP9 antibodyis an inhibitor of the activity of MMP9, and can be a specific inhibitorof MMP9. In particular, the MMP9 binding proteins disclosed herein willbe useful for inhibition of MMP9 while allowing normal function ofother, related matrix metalloproteinases. “An inhibitor of MMP” or“inhibitor of MMP9 activity” can be an antibody or an antigen bindingfragment thereof that directly or indirectly inhibits activity of MMP9,including but not limited to enzymatic processing, inhibiting action ofMMP9 on it substrate (e.g., by inhibiting substrate binding, substratecleavage, and the like), and the like.

The present disclosure also provides MMP9 binding proteins thatspecifically bind to non-mouse MMP9, such as human MMP9, Cynomolgusmonkey MMP9, and rat MMP9.

The present disclosure also provides MMP9 binding proteins (e.g.,anti-MMP9 antibodies and functional fragments thereof) that act asnon-competitive inhibitors. A “non-competitive inhibitor” refers to aninhibitor binds at site away from substrate binding site of an enzyme,and thus can bind the enzyme and effect inhibitory activity regardlessof whether or not the enzyme is bound to its substrate, Suchnon-competitive inhibitors can, for example, provide for a level ofinhibition that can be substantially independent of substrateconcentration.

MMP9 binding proteins (e.g., antibodies and functional fragmentsthereof) of the present disclosure include those that bind MMP9,particularly human MMP9, and having a heavy chain polypeptide (orfunctional fragment thereof) that has at least about 80%, 85%, 90%, 95%or more amino acid sequence identity to a heavy chain polypeptidedisclosed herein.

MMP9 binding proteins (e.g., antibodies and functional fragmentsthereof) of the present disclosure include those that bind MMP9,particularly human MMP9, and having a light polypeptide (or functionalfragment thereof) that has at least about 80%, 85%, 90%, 95% or moreamino acid sequence identity to a heavy chain polypeptide disclosedherein.

MMP9 binding proteins (e.g., antibodies and functional fragmentsthereof) of the present disclosure include those that bind MMP9,particularly human MMP9, and have a heavy chain polypeptide (orfunctional fragment thereof) having the complementarity determiningregions (“CDRs”) of heavy chain polypeptide and the CDRs of a lightchain polypeptide (or functional fragment thereof) as disclosed herein.

“Homology” or “identity” or “similarity” as used herein in the contextof nucleic acids and polypeptides refers to the relationship between twopolypeptides or two nucleic acid molecules based on an alignment of theamino acid sequences or nucleic acid sequences, respectively. Homologyand identity can each be determined by comparing a position in eachsequence which may be aligned for purposes of comparison. When anequivalent position in the compared sequences is occupied by the samebase or amino acid, then the molecules are identical at that position;when the equivalent site occupied by the same or a similar amino acidresidue (e.g., similar in steric and/or electronic nature), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of identical or similar amino acids atpositions shared by the compared sequences. In comparing two sequences,the absence of residues (amino acids or nucleic acids) or presence ofextra residues also decreases the identity and homology/similarity.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Sequences are generallyaligned for maximum correspondence over a designated region, e.g., aregion at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or moreamino acids or nucleotides in length, and can be up to the full-lengthof the reference amino acid or nucleotide. For sequence comparison,typically one sequence acts as a reference sequence, to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are input into a computer program, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Examples of algorithms that are suitable for determining percentsequence identity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 andAltschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov).Further exemplary algorithms include ClustalW (Higgins D., et al. (1994)Nucleic Acids Res 22: 4673-4680), available atwww.ebi.ac.uk/Tools/clustalw/index.html.

Residue positions which are not identical can differ by conservativeamino acid substitutions. Conservative amino acid substitutions refer tothe interchangeability of residues having similar side chains. Forexample, a group of amino acids having aliphatic side chains is glycine,alanine, valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.

Sequence identity between two nucleic acids can also be described interms of hybridization of two molecules to each other under stringentconditions. The hybridization conditions are selected following standardmethods in the art (see, for example, Sambrook, et al., MolecularCloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,N.Y.). An example of stringent hybridization conditions is hybridizationat 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodiumcitrate). Another example of stringent hybridization conditions isovernight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. Stringent hybridization conditions are hybridizationconditions that are at least as stringent as the above representativeconditions, where conditions are considered to be at least as stringentif they are at least about 80% as stringent, typically at least 90% asstringent as the above specific stringent conditions.

Accordingly, the present disclosure provides, for example, antibodies orantigen binding fragments thereof, comprising a heavy chain variableregion polypeptide having at least 80%, 85%, 90%, 95%, or greater aminoacid sequence identity to an amino acid sequence of a heavy chainvariable region described herein (e.g., SEQ ID NOS:1 or 5-8), and avariable light chain polypeptide having at least 80%, 85%, 90%, 95%, orgreater amino acid sequence identity to an amino acid sequence of alight chain polypeptide as set forth herein (e.g., SEQ ID NOS:2 or9-12).

Examples of anti-MMP9 antibodies of the present disclosure are describedin more detail below.

Antibodies

MMP9 binding proteins including antibodies and functional fragmentsthereof. As used herein, the term “antibody” means an isolated orrecombinant polypeptide binding agent that comprises peptide sequences(e.g., variable region sequences) that specifically bind an antigenicepitope. The term is used in its broadest sense and specifically coversmonoclonal antibodies (including full-length monoclonal antibodies),polyclonal antibodies, human antibodies, humanized antibodies, chimericantibodies, nanobodies, diabodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments including but not limitedto Fv, scFv, Fab, Fab′ F(ab′)₂ and Fab₂, so long as they exhibit thedesired biological activity. The term “human antibody” refers toantibodies containing sequences of human origin, except for possiblenon-human CDR regions, and does not imply that the full structure of animmunoglobulin molecule be present, only that the antibody has minimalimmunogenic effect in a human (i.e., does not induce the production ofantibodies to itself).

An “antibody fragment” comprises a portion of a full-length antibody,for example, the antigen binding or variable region of a full-lengthantibody. Such antibody fragments may also be referred to herein as“functional fragments: or “antigen-binding fragments”. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies (Zapata et al. (1995) Protein Eng.8(10):1057-1062); single-chain antibody molecules; and multispecificantibodies formed from antibody fragments. Papain digestion ofantibodies produces two identical antigen-binding fragments, called“Fab” fragments, each with a single antigen-binding site, and a residual“Fc” fragment, a designation reflecting the ability to crystallizereadily. Pepsin treatment yields an F(ab′)₂ fragment that has twoantigen combining sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the threecomplementarity-determining regions (CDRs) of each variable domaininteract to define an antigen-binding site on the surface of theV_(H)-V_(L) dimer. Collectively, the six CDRs confer antigen-bindingspecificity to the antibody. However, even a single variable domain (oran isolated V_(H) or V_(L) region comprising only three of the six CDRsspecific for an antigen) has the ability to recognize and bind antigen,although generally at a lower affinity than does the entire F_(v)fragment.

The “F_(ab)” fragment also contains, in addition to heavy and lightchain variable regions, the constant domain of the light chain and thefirst constant domain (CHO of the heavy chain. Fab fragments wereoriginally observed following papain digestion of an antibody. Fab′fragments differ from Fab fragments in that F(ab′) fragments containseveral additional residues at the carboxy terminus of the heavy chainCH₁ domain, including one or more cysteines from the antibody hingeregion. F(ab′)₂ fragments contain two Fab fragments joined, near thehinge region, by disulfide bonds, and were originally observed followingpepsin digestion of an antibody. Fab′-SH is the designation herein forFab′ fragments in which the cysteine residue(s) of the constant domainsbear a free thiol group. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to five majorclasses: IgA, IgD, IgE, IgG, and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise theV_(H) and V_(L) domains of antibody, wherein these domains are presentin a single polypeptide chain. In some embodiments, the Fv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains, which enables the sFv to form the desired structure for antigenbinding. For a review of sFv, see Pluckthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113 (Rosenburg and Moore eds.)Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain, thereby creating two antigen-binding sites. Diabodies areadditionally described, for example, in EP 404,097; WO 93/11161 andHollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment. Componentsof its natural environment may include enzymes, hormones, and otherproteinaceous or nonproteinaceous solutes. In some embodiments, anisolated antibody is purified (1) to greater than 95% by weight ofantibody as determined by the Lowry method, for example, more than 99%by weight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence, e.g., by use of a spinningcup sequenator, or (3) to homogeneity by gel electrophoresis (e.g.,SDS-PAGE) under reducing or nonreducing conditions, with detection byCoomassie blue or silver stain. The term “isolated antibody” includes anantibody in situ within recombinant cells, since at least one componentof the antibody's natural environment will not be present. In certainembodiments, isolated antibody is prepared by at least one purificationstep.

As used herein, “immunoreactive” refers to antibodies or fragmentsthereof that are specific to a sequence of amino acid residues (“bindingsite” or “epitope”), yet if are cross-reactive to otherpeptides/proteins, are not toxic at the levels at which they areformulated for administration to human use. “Epitope” refers to thatportion of an antigen capable of forming a binding interaction with anantibody or antigen binding fragment thereof. An epitope can be a linearpeptide sequence (i.e., “continuous”) or can be composed ofnoncontiguous amino acid sequences (i.e., “conformational” or“discontinuous”). The term “preferentially binds” means that the bindingagent binds to the binding site with greater affinity than it bindsunrelated amino acid sequences.

Anti-MMP9 antibodies can be described in terms of the CDRs of the heavyand light chains. As used herein, the term “CDR” or “complementaritydetermining region” is intended to mean the non-contiguous antigencombining sites found within the variable region of both heavy and lightchain polypeptides. These particular regions have been described byKabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S.Dept. of Health and Human Services, “Sequences of proteins ofimmunological interest” (1991); by Chothia et al., J. Mol. Biol.196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745(1996), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orgrafted antibodies or variants thereof is intended to be within thescope of the term as defined and used herein. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison.

TABLE 1 CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-3526-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L)CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

As used herein, the term “framework” when used in reference to anantibody variable region is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally a discontinuous amino acidsequence between about 100-120 amino acids in length but is intended toreference only those amino acids outside of the CDRs. As used herein,the term “framework region” is intended to mean each domain of theframework that is separated by the CDRs.

In some embodiments, an antibody is a humanized antibody or a humanantibody. Humanized antibodies include human immununoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. Thus, humanized forms ofnon-human (e.g., murine) antibodies are chimeric immunoglobulins whichcontain minimal sequence derived from non-human immunoglobulin. Thenon-human sequences are located primarily in the variable regions,particularly in the complementarity-determining regions (CDRs). In someembodiments, Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Humanized antibodies canalso comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences. In certain embodiments,a humanized antibody comprises substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDRs correspond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. For the purposes of the presentdisclosure, humanized antibodies can also include immunoglobulinfragments, such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-bindingsubsequences of antibodies.

The humanized antibody can also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. See, for example, Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta(1992) Curr. Op. Struct. Biol. 2:593-596.

Methods for humanizing non-human antibodies are known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” or “donor”residues, which are typically obtained from an “import” or “donor”variable domain. For example, humanization can be performed essentiallyaccording to the method of Winter and co-workers, by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody. See, for example, Jones et al., supra; Riechmann et al., supraand Verhoeyen et al. (1988) Science 239:1534-1536. Accordingly, such“humanized” antibodies include chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In certain embodiments, humanized antibodies arehuman antibodies in which some CDR residues and optionally someframework region residues are substituted by residues from analogoussites in rodent antibodies (e.g., murine monoclonal antibodies).

Human antibodies can also be produced, for example, by using phagedisplay libraries. Hoogenboom et al. (1991) J. Mol. Biol, 227:381; Markset al. (1991) J. Mol. Biol. 222:581. Other methods for preparing humanmonoclonal antibodies are described by Cole et al. (1985) “MonoclonalAntibodies and Cancer Therapy,” Alan R. Liss, p. 77 and Boerner et al.(1991) J. Immunol. 147:86-95.

Human antibodies can be made by introducing human immunoglobulin lociinto transgenic animals (e.g., mice) in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponimmunological challenge, human antibody production is observed, whichclosely resembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al. (1992) Bio/Technology 10:779-783(1992); Lonberg et al. (1994) Nature 368: 856-859; Morrison (1994)Nature 368:812-813; Fishwald et al. (1996) Nature Biotechnology14:845-851; Neuberger (1996) Nature Biotechnology 14:826; and Lonberg etal. (1995) Intern. Rev. Immunol. 13:65-93.

Antibodies can be affinity matured using known selection and/ormutagenesis methods as described above. In some embodiments, affinitymatured antibodies have an affinity which is five times or more, tentimes or more, twenty times or more, or thirty times or more than thatof the starting antibody (generally murine, rabbit, chicken, humanizedor human) from which the matured antibody is prepared.

An antibody can also be a bispecific antibody. Bispecific antibodies aremonoclonal, and may be human or humanized antibodies that have bindingspecificities for at least two different antigens. In the present case,the two different binding specificities can be directed to two differentMMPs, or to two different epitopes on a single MMP (e.g., MMP9).

An antibody as disclosed herein can also be an immunoconjugate. Suchimmunoconjugates comprise an antibody (e.g., to MMP9) conjugated to asecond molecule, such as a reporter An immunoconjugate can also comprisean antibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, a toxin (e.g., an enzymatically active toxin of bacterial,fungal, plant, or animal origin, or fragments thereof), or a radioactiveisotope (i.e., a radioconjugate).

An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope withoutsubstantially binding to any other polypeptide or polypeptide epitope.In some embodiments, an antibody of the present disclosure specificallybinds to human MMP9 with a dissociation constant (K_(d)) equal to orlower than 100 nM, optionally lower than 10 nM, optionally lower than 1nM, optionally lower than 0.5 nM, optionally lower than 0.1 nM,optionally lower than 0.01 nM, or optionally lower than 0.005 nM; in theform of monoclonal antibody, scFv, Fab, or other form of antibodymeasured at a temperature of about 4° C., 25° C., 37° C. or 42° C.

In certain embodiments, an antibody of the present disclosure binds toone or more processing sites (e.g., sites of proteolytic cleavage) inMMP9, thereby effectively blocking processing of the proenzyme orpreproenzyme to the catalytically active enzyme, and thus reducing theproteolytic activity of the MMP9.

In certain embodiments, an antibody according to the present disclosurebinds to MMP9 with an affinity at least 2 times, at least 5 times, atleast 10 times, at least 25 times, at least 50 times, at least 100times, at least 500 times, or at least 1000 times greater than itsbinding affinity for another MMP. Binding affinity can be measured byany method known in the art and can be expressed as, for example,on-rate, off-rate, dissociation constant (K_(d)), equilibrium constant(K_(eq)) or any term in the art.

In certain embodiments, an antibody according to the present disclosureis a non-competitive inhibitor of the catalytic activity of MMP9. Incertain embodiments, an antibody according to the present disclosurebinds within the catalytic domain of MMP9. In additional embodiments, anantibody according to the present disclosure binds outside the catalyticdomain of MMP9.

The present disclosure also contemplates antibodies or antigen bindingfragments thereof, that compete with anti-MMP9 antibodies or antigenbinding fragments thereof described herein for binding to MMP9. Thus,the present disclosure contemplates anti-MMP9 antibodies, and functionalfragments thereof, that compete for binding with, for example, anantibody having a heavy chain polypeptide of any of SEQ ID NOS;1 or 5-8,a light chain polypeptide of SEQ ID NOS:2 or 9-12, or combinationsthereof. In one embodiment, the anti-MMP9 antibody, for functionalfragment thereof, competes for binding to human MMP9 with the antibodydescribed herein as AB0041.

MMP9 Sequence

The amino acid sequence of human MMP9 protein is as follows:

(SEQ ID NO: 27) MSLWQPLVLV LLVLGCCFAA PRQRQSTLVL FPGDLRTNLT DRQLAEEYLY 50 RYGYTRVAEM RGESKSLGPA LLLLQKQLSL PETGELDSAT LKAMRTPRCG 100VPDLGRFQTF EGDLKWHHHN ITYWIQNYSE DLPRAVIDDA FARAFALWSA 150VTPLTFTRVY SRDADIVIQF GVAEHGDGYP FDGKDGLLAH AFPPGPGIQG 200DAHFDDDELW SLGKGVVVPT RFGNADGAAC HFPFIFEGRS YSACTTDGRS 250DGLPWCSTTA NYDTDDRFGF CPSERLYTRD GNADGKPCQF PFIFQGQSYS 300ACTTDGRSDG YRWCATTANY DRDKLFGFCP TRADSTVMGG NSAGELCVFP 350FTFLGKEYST CTSEGRGDGR LWCATTSNFD SDKKWGFCPD QGYSLFLVAA 400HEFGHALGLD HSSVPEALMY PMYRFTEGPP LHKDDVNGIR HLYGPRPEPE 450PRPPTTTTPQ PTAPPTVCPT GPPTVHPSER PTAGPTGPPS AGPTGPPTAG 500PSTATTVPLS PVDDACNVNI FDAIAEIGNQ LYLFKDGKYW RFSEGRGSRP 550QGPFLIADKW PALPRKLDSV FEEPLSKKLF FFSGRQVWVY TGASVLGPRR 600LDKLGLGADV AQVTGALRSG RGKMLLFSGR RLWRFDVKAQ MVDPRSASEV 650DRMFPGVPLD THDVFQYREK AYFCQDRFYW RVSSRSELNQ VDQVGYVTYD 700 ILQCPED 

Protein domains are shown schematically in FIG. 3 and are indicatedbelow:

Amino Acid # Feature  1-19 Signal Peptide 38-98 Peptidoglycan BindingDomain R98/C99 Propetide cleavage site (dependent on cleavage enzyme)112-445 Zn dependent metalloproteinase domain 223-271 Fibronectin typeII domain (gelatin binding domain) 281-329 Fibronectin type II domain(gelatin binding domain) 340-388 Fibronectin type II domain (gelatinbinding domain) 400-411 Zn binding region 521-565 Hemopexin-like domain567-608 Hemopexin-like domain 613-659 Hemopexin-like domain 661-704Hemopexin-like domain

The amino acid sequence of mature full-length human MMP9 (which is theamino acid sequence of the propolypeptide of SEQ ID NO:27 without thesignal peptide) is:

(SEQ ID NO: 28) PRQRQSTLVL FPGDLRTNLT DRQLAEEYLY RYGYTRVAEMRGESKSLGPA LLLLQKQLSL PETGELDSAT LKAMRTPRCGVPDLGRFQTF EGDLKWHHHN ITYWIQNYSE DLPRAVIDDAFARAFALWSA VTPLTFTRVY SRDADIVIQF GVAEHGDGYPFDGKDGLLAH AFPPGPGIQG DAHFDDDELW SLGKGVVVPTRFGNADGAAC HFPFIFEGRS YSACTTDGRS DGLPWCSTTANYDTDDRFGF CPSERLYTRD GNADGKPCQF PFIFQGQSYSACTTDGRSDG YRWCATTANY DRDKLFGFCP TRADSTVMGGNSAGELCVFP FTFLGKEYST CTSEGRGDGR LWCATTSNFDSDKKWGFCPD QGYSLFLVAA HEFGHALGLD HSSVPEALMYPMYRFTEGPP LHKDDVNGIR HLYGPRPEPE PRPPTTTTPQPTAPPTVCPT GPPTVHPSER PTAGPTGPPS AGPTGPPTAGPSTATTVPLS PVDDACNVNI FDAIAEIGNQ LYLFKDGKYWRFSEGRGSRP QGPFLIADKW PALPRKLDSV FEEPLSKKLFFFSGRQVWVY TGASVLGPRR LDKLGLGADV AQVTGALRSGRGKMLLFSGR RLWRFDVKAQ MVDPRSASEV DRMFPGVPLDTHDVFQYREK AYFCQDRFYW RVSSRSELNQ VDQVGYVTYD ILQCPEDwhere the amino acid sequence of the signal peptide is MSLWQPLVLVLLVLGCCFAA (SEQ ID NO:29).

The present disclosure contemplate MMP9 binding proteins that bind anyportion of MMP9, e.g., human MMP9, with MMP9 binding proteins thatpreferentially bind MMP9 relative to other MMPs being of particularinterest.

Anti-MMP9 antibodies, and functional fragments thereof, can be generatedaccordingly to methods well known in the art. Examples of anti-MMP9antibodies are provided below.

Mouse Monoclonal Anti-MMP9

A mouse monoclonal antibody to human MMP9 was obtained as described inExample 1. This antibody contains a mouse IgG2b heavy chain and a mousekappa light chain, and is denoted AB0041.

The amino acid sequence of the AB0041 heavy chain is as follows:

(SEQ ID NO: 1) MAVLVLFLCLVAFPSCVLSQVQLKESGPGLVAPSQSLSITCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTGGTTNYNSALMSRLSISKDDSKSQVFLKMNSLQTDDTAIYYCARYYYGMDYWGQGTSVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

The signal sequence is underlined, and the sequence of the IgG2bconstant region is presented italics.

The amino acid sequence of the AB0041 light chain is as follows:

(SEQ ID NO: 2) MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVRNTVAWYQQKTGQSPKLLIYSSSYRNTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYFCQQHYITPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

The signal sequence is underlined, and the sequence of the kappaconstant region is presented in italics.

The following amino acid sequence comprises the framework regions andcomplementarity-determining regions (CDRs) of the variable region of theIgG2b heavy chain of AB0041 (with CDRs underlined):

(SEQ ID NO: 3)QVQLKESGPGLVAPSQSLSITCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTGGTTNYNSALMSRLSISKDDSKSQVFLKMNSLQTDDTAIYYCARYYYGMDYWGQGTSVTVSS

The following amino acid sequence comprises the framework regions andcomplementarity-determining regions (CDRs) of the variable region of thekappa light chain of AB0041 (with CDRs underlined):

(SEQ ID NO: 4) DIVMTQSHKFMSTSVGDRVSITCKASQDVRNTVAWYQQKTGQSPKLLIYSSSYRNTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYFCQQHYITPYTF GGGTKLEIK

Heavy-Chain Variants

The amino acid sequences of the variable regions of the AB0041 heavy andlight chains were separately modified, by altering framework regionsequences in the heavy and light chain variable regions. The effect ofthese sequence alterations was to deplete the antibody of human T-cellepitopes, thereby reducing or abolishing its immunogenicity in humans(Antitope, Babraham, UK).

Four heavy-chain variants were constructed, in a human IgG4 heavy chainbackground containing a S241P amino acid change that stabilizes thehinge domain (Angal et al. (1993) Molec. Immunol. 30:105-108), and aredenoted VH1, VH2, VH3 and VH4. The amino acid sequences of theirframework regions and CDRs are as follows:

VH1 (SEQ ID NO: 5) QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTGGTTNYNSALMSRLTISKDDSKSTVYLKMNSLKTEDTAIYYCARY YYGMDYWGQGTSVTVSS VH2(SEQ ID NO: 6) QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTGGTTNYNSALMSRLTISKDDSKNTVYLKMNSLKTEDTAIYYCARY YYGMDYWGQGTLVTVSS VH3(SEQ ID NO: 7) QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTGGTTNYNSALMSRFTISKDDSKNTVYLKMNSLKTEDTAIYYCARY YYGMDYWGQGTLVTVSS VH4(SEQ ID NO: 8) QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTGGTTNYNSALMSRFTISKDDSKNTLYLKMNSLKTEDTAIYYCARY YYGMDYWGQGTLVTVSS

FIG. 1 shows an alignment of the amino acid sequences of the variableregions of the humanized heavy chains and indicates the differences inamino acid sequences in the framework regions among the four variants.

Light-Chain Variants

Four light-chain variants were constructed, in a human kappa chainbackground, and are denoted Vk1, Vk2, Vk3 and Vk4. The amino acidsequences of their framework regions and CDRs are as follows:

Vk1 (SEQ ID NO: 9) DIVMTQSPSFLSASVGDRVTITCKASQDVRNTVAWYQQKTGKAPKLLIYSSSYRNTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYFCQQHYITPYTF GGGTKVEIK Vk2(SEQ ID NO: 10) DIVMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSYRNTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYFCQQHYITPYTF GGGTKVEIK Vk3(SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSYRNTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQHYITPYTF GGGTKVEIK Vk4(SEQ ID NO: 12) DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSYRNTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYITPYTF GGGTKVEIK

FIG. 2 shows an alignment of the amino acid sequences of the variableregions of the humanized light chains and indicates the differences inamino acid sequences in the framework regions among the four variants.

The humanized heavy and light chains are combined in all possiblepair-wise combinations to generate a number of functional humanizedanti-MMP9 antibodies.

Additional heavy chain variable region amino acid sequences having 75%or more, 80% or more, 90% or more, 95% or more, or 99% or more homologyto the heavy chain variable region sequences disclosed herein are alsoprovided. Furthermore, additional light chain variable region amino acidsequences having 75% or more, 80% or more, 90% or more, 95% or more, or99% or more homology to the light chain variable region sequencesdisclosed herein are also provided.

Additional heavy chain variable region amino acid sequences having 75%or more, 80% or more, 90% or more, 95% or more, or 99% or more sequenceidentity to the heavy chain variable region sequences disclosed hereinare also provided. Furthermore, additional light chain variable regionamino acid sequences having 75% or more, 80% or more, 90% or more, 95%or more, or 99% or more sequence identity to the light chain variableregion sequences disclosed herein are also provided.

Complementarity-Determining Regions (CDRs)

The CDRs of the heavy chain of an anti-MMP9 antibody as disclosed hereinhave the following amino acid sequences:

(SEQ ID NO: 13) CDR1: GFSLLSYGVH (SEQ ID NO: 14) CDR2: VIWTGGTTNYNSALMS(SEQ ID NO: 15) CDR3: YYYGMDY

The CDRs of the light chain of an anti-MMP9 antibody as disclosed hereinhave the following amino acid sequences:

(SEQ ID NO: 16) CDR1: KASQDVRNTVA (SEQ ID NO: 17) CDR2: SSSYRNT(SEQ ID NO: 18) CDR3: QQHYITPYT

Nucleic Acids Encoding Anti-MMP9 Antibodies

The present disclosure provides nucleic acids encoding anti-MMP9antibodies and functional fragments thereof. Accordingly, the presentdisclosure provides an isolated polynucleotide (nucleic acid) encodingan antibody or antigen-binding fragment as described herein, vectorscontaining such polynucleotides, and host cells and expression systemsfor transcribing and translating such polynucleotides into polypeptides.

The present disclosure also contemplates constructs in the form ofplasmids, vectors, transcription or expression cassettes which compriseat least one polynucleotide as above.

The present disclosure also provides a recombinant host cell whichcomprises one or more constructs as above, as well as methods ofproduction of the antibody or antigen-binding fragments thereofdescribed herein which method comprises expression of nucleic acidencoding a heavy chain polypeptide and a light chain polypeptide (in thesame or different host cells, and from the same or different constructs)in a recombination host cell. Expression can be achieved by culturingunder appropriate conditions recombinant host cells containing thenucleic acid. Following production by expression, an antibody orantigen-binding fragment can be isolated and/or purified using anysuitable technique, then used as appropriate.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common bacterial hostis E. coli.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including operably linked promoter sequences,terminator sequences, polyadenylation sequences, enhancer sequences,marker genes and/or other sequences as appropriate. Vectors can beplasmids, viral e.g. ‘phage, or phagemid, as appropriate. For furtherdetails see, for example, Molecular Cloning: a Laboratory Manual: 2ndedition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Short Protocols inMolecular Biology, Second Edition, Ausubel et al. eds., John Wiley &Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. areincorporated herein by reference in their entirety.

The nucleic acid encoding a polypeptide of interest is integrated intothe genome of the host cell or can be maintained as a stable ortransient episomal element.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—can beused in these vectors to express the DNA sequences. For example, anucleic acid encoding a polypeptide of interest can be operably linkedto a promoter, and provided in an expression construct for use inmethods of production of recombinant MMP9 proteins or portions thereof.

Those of skill in the art are aware that nucleic acids encoding theantibody chains disclosed herein can be synthesized using standardknowledge and procedures in molecular biology.

Examples of nucleotide sequences encoding the heavy and light chainamino acid sequences disclosed herein, are as follows:

VH1: (SEQ ID NO: 19) CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGCCCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTTCTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCTCCAGGGAAGG GCCTGGAATG GCTGGGCGTG ATCTGGACCGGCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGCTGACCATCTCC AAGGACGACT CCAAGTCCAC CGTGTACCTGAAGATGAACT CCCTGAAAAC CGAGGACACC GCCATCTACTACTGCGCCCG GTACTACTAC GGCATGGACT ACTGGGGCCA GGGCACCTCC GTGACCGTGT CCTCAVH2: (SEQ ID NO: 20) CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGCCCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTTCTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCTCCAGGCAAAG GCCTGGAATG GCTGGGCGTG ATCTGGACCGGCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGCTGACCATCTCC AAGGACGACT CCAAGAACAC CGTGTACCTGAAGATGAACT CCCTGAAAAC CGAGGACACC GCCATCTACTACTGCGCCCG GTACTACTAC GGCATGGACT ACTGGGGCCA GGGCACCCTG GTCACCGTGT CCTCAVH3: (SEQ ID NO: 21) CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGCCCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTTCTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCTCCAGGCAAAG GCCTGGAATG GCTGGGCGTG ATCTGGACCGGCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGTTCACCATCTCC AAGGACGACT CCAAGAACAC CGTGTACCTGAAGATGAACT CCCTGAAAAC CGAGGACACC GCCATCTACTACTGCGCCCG GTACTACTAC GGCATGGACT ACTGGGGCCA GGGCACCCTG GTCACCGTGT CCTCAVH4 (SEQ ID NO: 22) CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGCCCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTTCTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCTCCAGGCAAAG GCCTGGAATG GCTGGGCGTG ATCTGGACCGGCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGTTCACCATCTCC AAGGACGACT CCAAGAACAC CCTGTACCTGAAGATGAACT CCCTGAAAAC CGAGGACACC GCCATCTACTACTGCGCCCG GTACTACTAC GGCATGGACT ACTGGGGCCA GGGCACCCTG GTCACCGTGT CGCAVk1: (SEQ ID NO: 23) GACATCGTGA TGACCCAGTC CCCCAGCTTC CTGTCCGCCTCCGTGGGCGA CAGAGTGACC ATCACATGCA AGGCCTCTCAGGACGTGCGG AACACCGTGG CCTGGTATCA GCAGAAAACCGGCAAGGCCC CCAAGCTGCT GATCTACTCC TCCTCCTACCGGAACACCGG CGTGCCCGAC CGGTTTACCG GCTCTGGCTCCGGCACCGAC TTTACCCTGA CCATCAGCTC CCTGCAGGCCGAGGACGTGG CCGTGTACTT CTGCCAGCAG CACTACATCACCCCCTACAC CTTCGGCGGA GGCACCAAGG TGGAAATAAA A Vk2: (SEQ ID NO: 24)GACATCGTGA TGACCCAGTC CCCCTCCAGC CTGTCCGCCTCTGTGGGCGA CAGAGTGACC ATCACATGCA AGGCCTCTCAGGACGTGCGG AACACCGTGG CCTGGTATCA GCAGAAGCCCGGCAAGGCCC CCAAGCTGCT GATCTACTCC TCCTCCTACCGGAACACCGG CGTGCCCGAC CGGTTTACCG GCTCTGGCTCCGGCACCGAC TTTACCCTGA CCATCAGCTC CCTGCAGGCCGAGGACGTGG CCGTGTACTT CTGCCAGCAG CACTACATCACCCCCTACAC CTTCGGCGGA GGCACCAAGG TGGAAATAAA A Vk3: (SEQ ID NO: 25)GACATCCAGA TGACCCAGTC CCCCTCCAGC CTGTCCGCCTCTGTGGGCGA CAGAGTGACC ATCACATGCA AGGCCTCCCAGGACGTGCGG AACACCGTGG CCTGGTATCA GCAGAAGCCCGGCAAGGCCC CCAAGCTGCT GATCTACTCC TCCTCCTACCGGAACACCGG CGTGCCCGAC CGGTTCTCTG GCTCTGGAAGCGGCACCGAC TTTACCCTGA CCATCAGCTC CCTGCAGGCCGAGGACGTGG CCGTGTACTT CTGCCAGCAG CACTACATCACCCCCTACAC CTTCGGCGGA GGCACCAAGG TGGAAATAAA A Vk4: (SEQ ID NO: 26)GACATCCAGA TGACCCAGTC CCCCTCCAGC CTGTCCGCCTCTGTGGGCGA CAGAGTGACC ATCACATGCA AGGCCTCTCAGGACGTGCGG AACACCGTGG CCTGGTATCA GCAGAAGCCCGGCAAGGCCC CCAAGCTGCT GATCTACTCC TCCTCCTACCGGAACACCGG CGTGCCCGAC CGGTTCTCTG GCTCTGGAAGCGGCACCGAC TTTACCCTGA CCATCAGCTC CCTGCAGGCCGAGGACGTGG CCGTGTACTA CTGCCAGCAG CACTACATCACCCCCTACAC CTTCGGCGGA GGCACCAAGG TGGAAATAAA A

Because the structure of antibodies, including the juxtaposition of CDRsand framework regions in the variable region, the structure of frameworkregions and the structure of heavy- and light-chain constant regions, iswell-known in the art; it is well within the skill of the art to obtainrelated nucleic acids that encode anti-MMP-9 antibodies. Accordingly,polynucleotides comprising nucleic acid sequences having at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98% andat least 99% homology to any of the nucleotide sequences disclosedherein are also provided. Accordingly, polynucleotides comprisingnucleic acid sequences having at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 98% and at least 99% identity toany of the nucleotide sequences disclosed herein are also provided.

Pharmaceutical Compositions

MMP9 binding proteins, as well as nucleic acid (e.g., DNA or RNA)encoding MMP9 binding proteins, can be provided as a pharmaceuticalcomposition, e.g., combined with a pharmaceutically acceptable carrieror excipient. Such pharmaceutical compositions are useful for, forexample, administration to a subject in vivo or ex vivo, and fordiagnosing and/or treating a subject with the MMP9 binding proteins.

Pharmaceutically acceptable carriers are physiologically acceptable tothe administered patient and retain the therapeutic properties of theantibodies or peptides with which it is administered.Pharmaceutically-acceptable carriers and their formulations are andgenerally described in, for example, Remington' pharmaceutical Sciences(18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa. 1990).One exemplary pharmaceutical carrier is physiological saline. Eachcarrier is “pharmaceutically acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notsubstantially injurious to the patient.

Pharmaceutical compositions can be formulated to be compatible with aparticular route of administration, systemic or local. Thus,pharmaceutical compositions include carriers, diluents, or excipientssuitable for administration by various routes.

Pharmaceutical compositions can include pharmaceutically acceptableadditives. Examples of additives include, but are not limited to, asugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose,sucrose, galactose, dextran, dextrose, fructose, lactose and mixturesthereof. Pharmaceutically acceptable additives can be combined withpharmaceutically acceptable carriers and/or excipients such as dextrose.Additives also include surfactants such as polysorbate 20 or polysorbate80.

The formulation and delivery methods will generally be adapted accordingto the site and the disease to be treated. Exemplary formulationsinclude, but are not limited to, those suitable for parenteraladministration, e.g., intravenous, intra-arterial, intramuscular, orsubcutaneous administration.

Pharmaceutical compositions for parenteral delivery include, forexample, water, saline, phosphate buffered saline, Hank's solution,Ringer's solution, dextrose/saline, and glucose solutions. Theformulations can contain auxiliary substances to approximatephysiological conditions, such as buffering agents, tonicity adjustingagents, wetting agents, detergents and the like. Additives can alsoinclude additional active ingredients such as bactericidal agents, orstabilizers. For example, the solution can contain sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate or triethanolamine oleate. Additional parenteralformulations and methods are described in Bai (1997) J. Neuroimmunol.80:65 75; Warren (1997) J. Neurol. Sci. 152:31 38; and Tonegawa (1997)J. Exp. Med. 186:507 515. The parenteral preparation can be enclosed inampules, disposable syringes or multiple dose vials made of glass orplastic.

Pharmaceutical compositions for intradermal or subcutaneousadministration can include a sterile diluent, such as water, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid, glutathione orsodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose.

Pharmaceutical compositions for injection include aqueous solutions(where water soluble) or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor ELTM (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof.Fluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Antibacterial andantifungal agents include, for example, parabens, chlorobutanol, phenol,ascorbic acid and thimerosal. Isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, and sodium chloride may beincluded in the composition. The resulting solutions can be packaged foruse as is, or lyophilized; the lyophilized preparation can later becombined with a sterile solution prior to administration.

Pharmaceutically acceptable carriers can contain a compound thatstabilizes, increases or delays absorption or clearance. Such compoundsinclude, for example, carbohydrates, such as glucose, sucrose, ordextrans; low molecular weight proteins; compositions that reduce theclearance or hydrolysis of peptides; or excipients or other stabilizersand/or buffers. Agents that delay absorption include, for example,aluminum monostearate and gelatin. Detergents can also be used tostabilize or to increase or decrease the absorption of thepharmaceutical composition, including liposomal carriers. To protectfrom digestion the compound can be complexed with a composition torender it resistant to acidic and enzymatic hydrolysis, or the compoundcan be complexed in an appropriately resistant carrier such as aliposome. Means of protecting compounds from digestion are known in theart (see, e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J.Pharm. Pharmacol. 48:119 135; and U.S. Pat. No. 5,391,377, describinglipid compositions for oral delivery of therapeutic agents).

Compositions of the present invention can be combined with othertherapeutic moieties or imaging/diagnostic moieties as provided herein.Therapeutic moieties and/or imaging moieties can be provided as aseparate composition, or as a conjugated moiety present on an MMP9binding protein.

Formulations for in vivo administration are generally sterile. In oneembodiment, the pharmaceutical compositions are formulated to be free ofpyrogens such that they are acceptable for administration to humanpatients.

Various other pharmaceutical compositions and techniques for theirpreparation and use will be known to those of skill in the art in lightof the present disclosure. For a detailed listing of suitablepharmacological compositions and associated administrative techniquesone can refer to the detailed teachings herein, which can be furthersupplemented by texts such as Remington: The Science and Practice ofPharmacy 20th Ed. (Lippincott, Williams & Wilkins 2003).

Pharmaceutical compositions can be formulated based on the physicalcharacteristics of the patient/subject needing treatment, the route ofadministration, and the like. Such can be packaged in a suitablepharmaceutical package with appropriate labels for the distribution tohospitals and clinics wherein the label is for the indication oftreating a disorder as described herein in a subject. Medicaments can bepackaged as a single or multiple units. Instructions for the dosage andadministration of the pharmaceutical compositions of the presentinvention can be included with the pharmaceutical packages and kitsdescribed below.

Methods of Use

The MMP9 binding proteins of the present disclosure can be used in, forexample, methods of detection of MMP9 in a sample, methods of treatment(e.g., as in methods of inhibition of angiogenesis), and methods ofdiagnosis. Examples of methods of use are described below.

Methods of Treatment

Provided herein are methods of treating diseases and disordersassociated with MMP9 activity. Diseases and disorder include, but arenot limited to tumors (e.g., primary or metastatic) that express or aredisposed in a tissue which expresses MMP9.

As used herein, “treat” or “treatment” means stasis or a postponement ofdevelopment of the symptoms associated a disease or disorder describedherein. The terms further include ameliorating existing uncontrolled orunwanted symptoms, preventing additional symptoms, and ameliorating orpreventing the underlying metabolic causes of symptoms. Thus, the termsdenote that a beneficial result has been conferred on a mammaliansubject with a disease or symptom, or with the potential to develop suchdisease or symptom. A response is achieved when the patient experiencespartial or total alleviation, or reduction of signs or symptoms ofillness, and specifically includes, without limitation, prolongation ofsurvival. The expected progression-free survival times can be measuredin months to years, depending on prognostic factors including the numberof relapses, stage of disease, and other factors.

The present disclosure contemplates pharmaceutical compositions for usein connection with such methods. Compositions can be suitable foradministration locally or systemically by any suitable route.

In general, MMP9 binding proteins are administered in a therapeuticallyeffective amount, e.g., in an amount to effect inhibition of tumorgrowth in a subject and/or to inhibit metastasis.

As used herein, the term “therapeutically effective amount” or“effective amount” refers to an amount of a therapeutic agent that whenadministered alone or in combination with another therapeutic agent to asubject is effective to prevent or ameliorate the disease condition orthe progression of the disease. A therapeutically effective dose furtherrefers to that amount of the compound sufficient to result inamelioration of symptoms, e.g., treatment, healing, prevention oramelioration of the relevant medical condition, or an increase in rateof treatment, healing, prevention or amelioration of such conditions.When applied to an individual active ingredient administered alone, atherapeutically effective dose refers to that ingredient alone. Whenapplied to a combination, a therapeutically effective dose refers tocombined amounts of the active ingredients that result in thetherapeutic effect, whether administered in combination, serially orsimultaneously. For example, when in vivo administration of an anti-MMP9antibody is employed, normal dosage amounts can vary from about 10 ng/kgto up to 100 mg/kg of mammal body weight or more per day, preferablyabout 1 μg/kg/day to 50 mg/kg/day, optionally about 100 μg/kg/day to 20mg/kg/day, 500 μg/kg/day to 10 mg/kg/day, or 1 mg/kg/day to 10mg/kg/day, depending upon the route of administration.

The selected dosage regimen will depend upon a variety of factorsincluding the activity of the MMP9 binding protein, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular composition employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A clinician having ordinary skill in the art can readily determine andprescribe the effective amount (ED50) of the pharmaceutical compositionrequired. For example, the physician or veterinarian can start doses ofthe compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

As used herein, the term “subject” means mammalian subjects. Exemplarysubjects include, but are not limited to humans, monkeys, dogs, cats,mice, rats, cows, horses, goats and sheep. In some embodiments, thesubject has cancer and can be treated with the agent of the presentinvention as described below.

If needed, for cancer treatments, methods can further include surgicalremoval of the cancer and/or administration of an anti-cancer agent ortreatment in addition to an MMP9 binding protein. Administration of suchan anti-cancer agent or treatment can be concurrent with administrationof the compositions disclosed herein.

Methods of Detection of MMP9

The present disclosure also contemplates methods of detecting MMP9 in asubject, e.g., to detect tumor or tumor-associated tissue expressingMMP9. Thus, methods of diagnosing, monitoring, staging or detecting atumor having MMP9 activity are provided.

Samples from an individual suspected of having a tumor associated withMMP9 expression can be collected and analyzed by detecting the presenceor absence of binding of an MMP9 binding protein. This analysis can beperformed prior to the initiation of treatment using an MMP9 bindingprotein as described herein, or can be done as part of monitoring ofprogress of cancer treatment. Such diagnostic analysis can be performedusing any sample, including but not limited to tissue, cells isolatedfrom such tissues, and the like. Tissue samples include, for example,formalin-fixed or frozen tissue sections.

Any suitable method for detection and analysis of MMP9 be employed.Various diagnostic assay techniques known in the art can be adapted forsuch purpose, such as competitive binding assays, direct or indirectsandwich assays and immunoprecipitation assays conducted in eitherheterogeneous or homogeneous phases.

MMP9 binding proteins for use in detection methods can be labeled with adetectable moiety. The detectable moiety directly or indirectly producesa detectable signal. For example, the detectable moiety can be any ofthose described herein such as, for example, a radioisotope, such as 3H,14C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, suchas fluorescein isothiocyanate (FITC), Texas red, cyanin, photocyan,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,β-galactosidase or horseradish peroxidase.

Detection can be accomplished by contacting a sample under conditionssuitable for MMP9 binding protein binding to MMP9, and assessing thepresence (e.g., level) or absence of MMP9 binding protein-MMP9complexes. A level of MMP9 in the sample in comparison with a level of areference sample can indicate the presence of a tumor ortumor-associated tissues having MMP9 activity. The reference sample canbe a sample taken from the subject at an earlier time point or a samplefrom another individual.

EXAMPLES Example 1 Preparation of Antibodies to Human MMP-9

The full-length human MMP9 protein without a signal peptide, which isSEQ ID NO. 28 was used to immunize mice. Spleen cells from immunizedmice were fused with myeloma cells to generate a hybridoma library.Monoclonal cultures were prepared and screened to identify culturesexpressing an anti-MMP9 monoclonal antibody.

Antibody (AB0041) was purified from one of the cultures andcharacterized. The antibody contained an IgG2b heavy chain and a kappalight chain. Characterization included testing for the binding of AB0041to other human MMPs and to MMP9 proteins from other species, includingcynomolgus monkey, rat and mouse. It was found that the AB0041 antibodybound strongly to human and cynomolgus MMP9, that it bound less stronglyto rat MMP9, and that it did not bind to murine MMP9 or to many of thehuman non-MMP matrix metalloproteinases.

TABLE 2 Cross reactivity of AB0041 and AB0045. Dissociation constant(Kd) MMP Tested AB0045 AB0041 Human MMP1 >100 nM >100 nM Human MMP2 >100nM >100 nM Mouse MMP2 >100 nM >100 nM Human MMP3 >100 nM >100 nM HumanMMP7 >100 nM >100 nM Human MMP8 >100 nM >100 nM Human MMP9 0.168 ± 0.117nM 0.133 ± 0.030 nM Cynomolgus monkey 0.082 ± 0.022 nM 0.145 ± 0.16 nM MMP9 Mouse MMP9 >100 nM >100 nM Rat MMP9 0.311 ± 0.017 nM 0.332 ± 0.022nM Human MMP10 >100 nM >100 nM Human MMP12 >100 nM >100 nM HumanMMP13 >100 nM >100 nM

Additional characterization included assaying the binding of theantibody to mouse MMP9 in which certain amino acids were altered to moreclosely correspond to the human MMP9 sequence. In addition, the humanMMP9 protein was mutagenized, and the various mutants tested for theirability to be bound by the antibody, to determine amino acids importantfor antibody binding and thereby define the therapeutic epitope. Thisanalysis identified an arginine residue at position 162 of the MMP9amino acid sequence (R162) as important for antibody binding. Otheramino acid residues in MMP9 that are important for binding of the AB0041antibody include E111, D113, and 1198. Recent crystal structure of MMP9showed that E111, D113, R162, and 1198 were grouped near each otheraround a Ca2+ ion binding pocket of MMP9. Without binding to anyspecific scientific theory, AB0041 may bind to the region on MMP9wherein these residues are located. Alternatively, these MMP9 residuesmay have direct contact with AB0041.

In an enzymatic assay for MMP9, the AB0041 antibody was found to act asa non-competitive inhibitor.

Example 2 Humanization of Antibodies to Human MMP9

The amino acid sequences of the heavy chain and light chain of the mouseAB0041 antibody were altered at certain locations in the framework(i.e., non-CDR) portion of their variable regions to generate proteinsthat are less immunogenic in humans. These amino acid sequence changeswere shown in FIGS. 1 and 2. The cross-reactivity of the humanizedantibody (referred to as AB0045) is shown in Table 2 above.

What is claimed is:
 1. An isolated matrix metalloproteinase 9 (MMP9)binding protein, comprising: an immunoglobulin heavy chain polypeptide,or functional fragment thereof, and an immunoglobulin light chainpolypeptide, or functional fragment thereof, wherein the MMP9 bindingprotein specifically binds to an to epitope of human MMP9 comprisingamino acid residues R162, E111, D113, and I198.